The invention relates to a novel gene encoding a protein that associates with human telomeres, and to compounds that interact with telomeric DNA binding proteins, thereby acting to extend or shorten telomere length.
Telomeres are DNA-protein structures that cap the ends of linear eukaryotic chromosomes. Telomeres consist of several thousand copies of a repetitive DNA sequence (TTAGGG in vertebrates), and an unknown number of proteins. The telomeric nucleic acid-protein structure is essential for preventing chromosome end-to-end fusions and, thus, for maintaining genomic stability (Zakian, 1989; Blackburn, 1991). Telomeres can also influence gene expression. In lower eukaryotes, genes located near telomeres are silenced, and proteins that mediate this silencing can alter gene expression at non-telomeric loci (Aparicio et al., 1991; Brachmann et al., 1995; Marchand et al., 1996). In higher eukaryotes, telomere shortening causes striking changes in cell phenotype (Campisi, 1997). The ability of telomeres to prevent genomic instability and alter gene expression depends on their length and the proteins that associate with them.
The human germ line and early embryonic cells maintain an average telomere or terminal restriction fragment (TRF) length of 15-20 kb. This length is maintained in part by telomerase, a ribonucleoprotein reverse transcriptase (Greider, 1996; Lingner and Cech, 1998; Nugent and Lundblad, 1998). Most human cells do not express telomerase. Because DNA replication is bidirectional, initiated from a labile primer and catalyzed by a unidirectional polymerase, each cell cycle leaves unduplicated 50-200 bp at the 3xe2x80x2 terminus (Levy et al., 1992). Telomerase uses this 3xe2x80x2 overhang to add back single-stranded telomeric repeats, but proliferating cells that lack telomerase lose telomeric DNA. After 50-80 divisions, most human cells have TRFs of only 4-7 kb, at which point they enter an irreversible state of arrested growth and altered function termed replicative senescence (Harley et al., 1990; Shay and Wright, 1991; Dimri et al., 1995; Campisi et al., 1996). Replicative senescence is an important tumor suppressive mechanism, and the accumulation of dysfunctional senescent cells may contribute to certain age-related pathologies (Sager, 1991; Harley and Villeponteau, 1995; Campisi, 1996, 1997; Yeager et al., 1998).
Ectopic expression of telomerase prevents telomere erosion and senescence in some, but not all, human cells (Bodnar et al., 1998; Vaziri and Benchimol, 1998; Kiyono et al., 1998). In addition, viral oncoproteins that inactivate the cellular tumor suppressors p53 and pRb delay or prevent senescence (Weinberg, 1991). Such proteins do not, however, prevent telomere shortening. Human cells lacking p53 and pRb function can proliferate until the telomeres become very short ( less than 2 kb) and the genome unstable, whereupon cells with an indefinite or immortal replicative life span may emerge (Shay and Wright, 1991). Immortalization renders cells highly susceptibility to tumorigenic transformation (Sager, 1991), but tumor cells cannot survive unless they acquire a means to maintain their telomeres. The most common means is induction of telomerase (Kim et al., 1994), but recombination can also prevent telomere loss (Bryan et al., 1997). In addition to telomerase, telomere length is regulated by exonuclease activity, and telomere-associated proteins that may determine whether and how telomerase gains access to the 3xe2x80x2 terminus (Greider, 1996; Shore, 1997; Lingner and Cech, 1998).
Lower eukaryotes such as Saccharomyces cerevisiae maintain telomeres by a balance between elongation by telomerase and shortening by exonuclease activity. This equilibrium is controlled in part by Rap1, a double-stranded telomeric DNA binding protein. Rap1 negatively regulates telomere length, and maintains chromosome stability and telomeric silencing (Conrad et al., 1990; Kyrion et al., 1992). At least two Rap1 binding proteins, Rif1p and Rif2p, are important for Rap1p function (Hardy et al., 1992; Wotton and Shore, 1997). In addition, Rap1 associates with components of the Sir complex, which regulate silencing at telomeric and non-telomeric loci (Cockell et al., 1995; Marchand et al., 1996). Yeast proteins that associate with the telomeric 3xe2x80x2 overhang have also been identified, two of which, Cdc13 and its binding protein Stn1, negatively regulate telomere length (Grandin et al., 1997; Nugent et al., 1996).
Three genes encoding human telomere-associated proteins have been cloned. Trf1 (Chong et al., 1995), the first such gene, may be a functional homologue of Rap1. Trf1 and its alternately spliced form Pin2 (Shen et al., 1997) bind double-stranded telomeric DNA and negatively regulate telomere length (van Steensel and de Lange, 1997). Trf1 also promotes parallel pairing of telomeric DNA tracts (Griffith et al., 1998). Trf2 is architecturally similar to Trf1, prevents chromosome fusions (van Steensel et al., 1998). A third protein, tankyrase, was recently identified as a Trf1-interacting protein and shown to have poly-ADP ribosylase activity (Smith et al., 1998).
The identification of proteins that modulate telomere length and telomerase activity provides important tools for the diagnosis and treatment of human disease. Compounds that inhibit telomerase activity can be used to treat cancer, as cancer cells express telomerase activity and normal human somatic cells do not express telomerase activity at biologically relevant levels (i.e., at levels sufficient to maintain telomere length over many cell divisions). There is a need for compounds that act as telomerase inhibitors and for compositions and methods for treating cancer and other diseases in which telomerase activity is present abnormally. Certain age-related disorders may be treated by lengthening telomeres (e.g., improvements in wound healing, immune response).
Accordingly, it is an object of the present invention to provide a novel recombinant protein (termed herein xe2x80x9cTin2xe2x80x9d) that is associated with other telomere binding proteins.
It is further an object of the present invention to provide a polynucleotide useful in the production of this protein.
It is another object of the invention to provide materials and methods useful in the evaluation of the ability of test substances to modulate telomere length.
It is also an object of the present invention to provide materials and methods which can be useful in evaluating the telomere status of a subject cell.
It is another object of the invention to provide antibodies to Tin2, said antibodies being useful in measuring the expression of Tin2.
It is an additional object of the present invention to provide nucleic acids in the form of Tin2 probes for detecting the presence of the,Tin2 gene and/or Tin2 transcription.
It is also an object of the present invention to provide a composition and method for the promotion clustering of telomeric DNA tracts.
The present invention comprises a protein, Tin2, that associates with mammalian telomeres. Tin2 interacts with Trf1, and negatively regulates telomere length. Tin2 does not directly bind DNA, but mediates formation of a Tin2-Trf1-telomeric DNA multiplex that limits telomerase access to the telomere. Tin2 also aligns telomeric DNA tracts.
The present invention provides polynucleotide (specifically cDNA) sequences that encode a novel Trf1 binding protein having approximately 354 amino acids. The encoded wild-type Tin2 protein negatively regulates telomere length. In addition, mutant DNA and proteins are provided that contain deletions in the wild-type structures. These mutant proteins can induce elongation of telomeres in telomerase-positive cells.
Using the DNA sequences for human Tin2 provided herein, one can also obtain nucleic acid probes specific for the Tin2 gene or the Tin2 mRNA. These probes can be used to ascertain the status of a test cell as to (a) whether it possesses the Tin2 gene or (2) whether and how much it is expressing Tin2.
Using the DNA sequences for human Tin2 provided herein, one can also obtain related genes from other species or Tin2 homologs, such as the mouse.
Furthermore, antibodies are provided herein which are specific to Tin2. Polyclonal antibodies have been produced in rabbits, and monoclonal antibodies can be produced by known methods. These antibodies can be used to ascertain the Tin2 localization in a test cell, as well as the expression of Tin2 in a test cell.
Also provided are assay methodologies that utilize the Tin2 protein, DNA or antibody herein provided to screen potential therapeutic compounds that inhibit or increase (mimic) Tin2 or telomerase function. It is believed that since the Trf1-Tin2 complex inhibits telomerase function, a test compound that inhibits the binding of Tin2 to Trf will increase telomerase activity. A compound that mimics wild type Tin2 by preventing association between telomerase and the Trf1 complex would inhibit telomerase activity.
Finally, the present Tin2 DNA and protein could be added to cells to provide either decreased telomerase activity (wild type) or increased telomerase activity (mutated). A specific mutation, truncation Tin2-13 is especially preferred to increase wild-type telomerase activity, i.e., telomere length. The Tin2 constructs of the present invention may be delivered to host cells by transfection with retroviral vectors package using a known packaging cell line. The coat protein is trophic for human and animal cells.